RESUMO
The immune response plays a critical role in the pathophysiology of SARS-CoV-2 infection ranging from protection to tissue damage and all occur in the development of acute respiratory distress syndrome (ARDS). ARDS patients display elevated levels of inflammatory cytokines and innate immune cells, and T and B cell lymphocytes have been implicated in this dysregulated immune response. Mast cells are abundant resident cells of the respiratory tract and are able to release different inflammatory mediators rapidly following stimulation. Recently, mast cells have been associated with tissue damage during viral infections, but their role in SARS-CoV-2 infection remains unclear. In this study, we examined the profile of mast cell activation markers in the serum of COVID-19 patients. We noticed that SARS-CoV-2-infected patients showed increased carboxypeptidase A3 (CPA3) and decreased serotonin levels in their serum when compared with symptomatic SARS-CoV-2-negative patients. CPA3 levels correlated with C-reactive protein, the number of circulating neutrophils, and quick SOFA. CPA3 in serum was a good biomarker for identifying severe COVID-19 patients, whereas serotonin was a good predictor of SARS-CoV-2 infection. In summary, our results show that serum CPA3 and serotonin levels are relevant biomarkers during SARS-CoV-2 infection. This suggests that mast cells and basophils are relevant players in the inflammatory response in COVID-19 and may represent targets for therapeutic intervention.
Assuntos
COVID-19/diagnóstico , Carboxipeptidases A/metabolismo , Mediadores da Inflamação/metabolismo , Inflamação/diagnóstico , Mastócitos/imunologia , SARS-CoV-2/isolamento & purificação , Serotonina/metabolismo , Biomarcadores/análise , COVID-19/complicações , COVID-19/metabolismo , COVID-19/virologia , Humanos , Inflamação/etiologia , Inflamação/metabolismo , Inflamação/patologia , Mastócitos/patologia , Índice de Gravidade de DoençaRESUMO
Carboxypeptidase A (CPA) is a metalloexopeptidase that catalyzes the hydrolysis of the peptide bonds that are adjacent to the C-terminal end of a polypeptide chain. The enzyme preferentially cleaves over C-terminal L-amino acids with aromatic or branched side chains. This is of main importance for food industry because it can be employed for manufacturing functional foods from different protein sources with reduced hydrophobic amino acid content for patients with deficiencies in the absorption or digestion of the corresponding amino acids. In that way, strategies for effective multipoint covalent immobilization of CPA metalloenzyme on chitosan beads have been developed. The study of the ability to produce several chemical modifications on chitosan molecules before, during and after its coagulation to form carrier beads lead in a protective effect of the polymer matrix. The chemical modification of chitosan through the use of an N-alkylation strategy produced the best derivatives. N-alkyl chitosan derivative beads with D-fructose presented values of 0.86 for immobilization yield, 314.6 IU g-1 bead for initial activity of biocatalyst and were 5675.64-fold more stable than the free enzyme at 55 °C. Results have shown that these derivatives would present a potential technological application in hydrolytic processes due to both their physical properties, such as low swelling capacity, reduced metal chelation ability and bulk mesoporosity, and increased operational stability when compared with soluble enzyme.
Assuntos
Carboxipeptidases A/química , Quitosana/química , Enzimas Imobilizadas/química , Biocatálise , Estabilidade Enzimática , Frutose/química , Temperatura AltaRESUMO
Carboxypeptidase A6 (CPA6) is an extracellular matrix metallocarboxypeptidase that modulates peptide and protein function by removal of hydrophobic C-terminal amino acids. Mutations in the human CPA6 gene that reduce enzymatic activity in the extracellular matrix are associated with febrile seizures, temporal lobe epilepsy, and juvenile myoclonic epilepsy. The characterization of these human mutations suggests a dominant mode of inheritance by haploinsufficiency through loss of function mutations, however the total number of humans with pathologic mutations in CPA6 identified to date remains small. To better understand the relationship between CPA6 and seizures we investigated the effects of morpholino knockdown of cpa6 mRNA in zebrafish (Danio rerio) larvae. Knockdown of cpa6 mRNA resulted in resistance to the effect of seizure-inducing drugs pentylenetetrazole and pilocarpine on swimming behaviors. Knockdown of cpa6 mRNA also reduced the levels of mRNAs encoding neuropeptide precursors (bdnf, npy, chga, pcsk1nl, tac1, nts, edn1), a neuropeptide processing enzyme (cpe), transcription factor (c-fos), and molecules implicated in glutamatergic signaling (grin1a and slc1a2b). Treatment of zebrafish embryos with 60 mM pilocarpine for 1 hour led to reductions in levels of many of the same mRNAs when measured 1 day after pilocarpine exposure, except for c-fos which was elevated 1 day after pilocarpine treatment. Pilocarpine treatment, like cpa6 knockdown, led to a reduced sensitivity to pentylenetetrazole when tested 1 day after pilocarpine treatment. Taken together, these results add to mounting evidence that peptidergic systems participate in the biological effects of seizure-inducing drugs, and are the first in vivo demonstration of the molecular and behavioral consequences of cpa6 insufficiency.
Assuntos
Carboxipeptidases A/genética , Técnicas de Silenciamento de Genes , Larva/enzimologia , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Animais , Convulsivantes/administração & dosagem , Mutação , Pilocarpina/administração & dosagem , RNA Mensageiro/genética , Transcrição Gênica , Peixe-Zebra/embriologiaRESUMO
Milk whey proteins are well known for their high biological value and versatile functional properties, characteristics that allow its wide use in the food and pharmaceutical industries. In this work, a 24 kDa protein from buffalo cheese whey was analyzed by mass spectrometry and presented homology with Bos taurus beta-lactoglobulin. In addition, the proteins present in buffalo cheese whey were hydrolyzed with pepsin and with different combinations of trypsin, chymotrypsin and carboxypeptidase-A. When the TNBS method was used the obtained hydrolysates presented DH of 55 and 62% for H1 and H2, respectively. Otherwise for the OPA method the DH was 27 and 43% for H1 and H2, respectively. The total antioxidant activities of the H1 and H2 samples with and without previous enzymatic hydrolysis, determined by DPPH using diphenyl-p-picrylhydrazyl radical, was 4.9 and 12 mM of Trolox equivalents (TE) for H2 and H2Dint, respectively. The increased concentrations for H1 and H2 samples were approximately 99% and 75%, respectively. The in vitro gastrointestinal digestion efficiency for the samples that were first hydrolyzed was higher compared with samples not submitted to previous hydrolysis. After in vitro gastrointestinal digestion, several amino acids were released in higher concentrations, and most of which were essential amino acids. These results suggest that buffalo cheese whey is a better source of bioavailable amino acids than bovine cheese whey.
Assuntos
Queijo/análise , Análise de Alimentos/métodos , Hidrolisados de Proteína/química , Proteínas do Soro do Leite/química , Soro do Leite/metabolismo , Aminoácidos/química , Animais , Antioxidantes/química , Compostos de Bifenilo/química , Búfalos , Carboxipeptidases A/química , Bovinos , Cromanos/química , Quimotripsina/química , Trato Gastrointestinal/metabolismo , Hidrólise , Lactoglobulinas/química , Lactose/química , Espectrometria de Massas , Peptídeos/química , Picratos/química , Tripsina/químicaRESUMO
A study was performed in order to understand the development of digestive enzymes during initial ontogeny of Cichlasoma trimaculatum, for which the activity of acidic and alkaline proteases, lipases, amylases and phosphatases was determined by means of biochemical and electrophoretic analysis. Our results showed that the activity of alkaline proteases, trypsin and chymotrypsin is present from day 6 after hatching (dah) during exogenous feeding with Artemia nauplii. The activities of carboxypeptidase A and leucine aminopeptidase are present from the first days, increasing at 6 dah and reaching their maximum activity at 9 dah while acid protease activity started at 9 dah. Furthermore, the lipase activity is detected on 6 dah and keeps increasing and decreasing on 17 dah. Amylase activity is detected on 3 dah, presenting fluctuations until 45 dah, where it reaches its maximum activity. Acid and alkaline phosphatases are detected from 3 dah and reach a maximum activity between 13 and 19 dah. The SDS-PAGE electrophoresis revealed six types of bands in the alkaline proteases, with molecular weight between 113.4 and 20.4 kDa. First three bands appear on 6 dah, but it is until 11 dah when all isoforms appear. Based on these results, it is considered that this species completes its digestive enzymatic machinery from day 9 after hatching, therefore is recommended to perform the transition from live feed to inert feed at 15 dah.
Assuntos
Aquicultura/métodos , Ciclídeos/crescimento & desenvolvimento , Sistema Digestório/enzimologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Fatores Etários , Amilases/metabolismo , Animais , Carboxipeptidases A/metabolismo , Quimotripsina/metabolismo , Ciclídeos/metabolismo , Sistema Digestório/crescimento & desenvolvimento , Eletroforese em Gel de Poliacrilamida , Leucil Aminopeptidase/metabolismo , Lipase/metabolismo , Peptídeo Hidrolases/metabolismo , Tripsina/metabolismoRESUMO
Tropical gar (Atractosteus tropicus) is an economically and socially important freshwater species from Southeastern Mexico, with a high aquaculture potential. With this in mind, the purpose of this study was to characterize the digestive proteases of tropical gar juveniles through biochemical and electrophoretic analyses. Twenty specimens with an average weight of 73.6 ± 12.7 g were used to obtain stomach and intestinal tissue from which multienzymatic extracts were prepared. The general activities of the acid and alkaline proteases were evaluated, as well as the specific activities of trypsin, chymotrypsin, leucine aminopeptidase and carboxypeptidase A. The effect of the pH and temperature on the proteases was also analyzed, together with the composition of the multienzymatic extracts using protease inhibitors and electrophoretic tests. Results showed that A. tropicus have a functional stomach in which protein hydrolysis starts with pepsin and which contains endo- and exopeptidases (trypsin, chymotrypsin, leucine aminopeptidase and carboxypeptidase A) and proteases that are resistant to high temperatures (45 and 55 °C for alkaline and acid proteases, respectively) and pH values. Using zymogram technique, we found two acid protease isoforms (0.35 and 0.71 rf) and five alkaline protease isoforms (83.7, 43.7, 27.5, 24.0 and 19.4 kDa), which decrease or disappear with the different inhibitors. Thus, this species is considered to be a carnivore capable of adapting to its environment by consuming different types of proteins from preys and also could adapt rapidly to consume a compound diet with different animal protein sources.
Assuntos
Digestão/fisiologia , Peixes/metabolismo , Trato Gastrointestinal/enzimologia , Peptídeo Hidrolases/metabolismo , Animais , Aquicultura/métodos , Carboxipeptidases A/metabolismo , Quimotripsina/metabolismo , Eletroforese/veterinária , Peixes/fisiologia , Concentração de Íons de Hidrogênio , Isoenzimas/metabolismo , Leucil Aminopeptidase/metabolismo , México , Estatísticas não Paramétricas , Temperatura , Tripsina/metabolismoRESUMO
Here we report the isolation of carboxypeptidases A1 and A2 (CPA1 and CPA2) from the rat mesenteric arterial bed perfusate, which were found to be identical with their pancreatic counterparts. Angiotensin (Ang) I, Ang II, Ang-(1-9) and Ang-(1-12) were differentially processed by these enzymes, worthy mentioning the peculiar CPA1-catalyzed conversion of Ang II to Ang-(1-7) and the CPA2-mediated formation of Ang I from Ang-(1-12). We detected gene transcripts for CPA1 and CPA2 in mesentery and other extrapancreatic tissues, indicating that these CPAs might play a role in the renin-angiotensin system in addition to their functions as digestive enzymes.
Assuntos
Angiotensina II/metabolismo , Angiotensina I/metabolismo , Carboxipeptidases A/genética , Carboxipeptidases A/metabolismo , Artérias Mesentéricas/enzimologia , Sequência de Aminoácidos , Angiotensinogênio , Angiotensinas/metabolismo , Animais , Sequência de Bases , Regulação Enzimológica da Expressão Gênica , Técnicas In Vitro , Cinética , Artérias Mesentéricas/metabolismo , Dados de Sequência Molecular , Especificidade de Órgãos , Fragmentos de Peptídeos/metabolismo , Perfusão , RatosRESUMO
UNLABELLED: This paper proposes a method to enzymatically treat poor-quality cocoa almonds (known as "slate") to ensure the formation of chocolate flavor precursors. The production of flavor precursors improves the quality of these almonds, which are usually responsible for the low quality of the liquor produced. Proteases and carboxypeptidases from different sources were tested under various conditions. The different treatments were evaluated by chemical analysis (hydrolysis efficiency) and sensory analysis of the treated material compared to good-quality cocoa almonds. The results show that it is possible, through the use of microbial enzymes, to generate the mixture of compounds that will release, after roasting, the characteristic chocolate flavor in poor-quality almonds. However, it is necessary to optimize the conditions of enzymatic treatment to obtain better results and thus establish a process that can be used for industrial purposes for manufacturing cocoa and chocolate. PRACTICAL APPLICATION: The basidiomycete Moniliophtora perniciosa is the causative agent of witches' broom disease (WBD) of the cocoa tree, whose seeds are the source of chocolate. It is the most important phytopathological problem of cocoa-producing areas of the American continent, and has decimated the Brazilian cocoa industry. In Bahia (Brazil), M. perniciosa was identified in 1989 and, as a consequence of its spreading, the annual production of cocoa almonds dropped from 450,000 to 90,000 tons within 12 y, reducing export values from an all-time high of about US$ 1 billion to 110 million. The high incidence of WBD incapacitates Brazil to produce enough cocoa almonds even for the internal market, leading the country to import low-quality cocoa almonds mainly from African countries. Our work proposes an enzymatic treatment to increase the quality of that cocoa almonds and, consequently, to improve the quality of the chocolate produced and consumed in the country.
Assuntos
Cacau/química , Enzimas/metabolismo , Manipulação de Alimentos/métodos , Prunus/química , Sementes/química , Paladar , África , Agaricales/patogenicidade , Brasil , Carboxipeptidases A/metabolismo , Pepsina A/metabolismo , Doenças das Plantas/microbiologiaRESUMO
This paper presents stable carboxypeptidase A (CPA)-glyoxyl derivatives, to be used in the controlled hydrolysis of proteins. They were produced after immobilizing-stabilizing CPA on cross-linked 6% agarose beads, activated with low and high concentrations of aldehyde groups, and different immobilization times. The CPA-glyoxyl derivatives were compared to other agarose derivatives, prepared using glutaraldehyde as activation reactant. The most stabilized CPA-glyoxyl derivative was produced using 48 h of immobilization time and high activation grade of the support. This derivative was approximately 260-fold more stable than the soluble enzyme and presented approximately 42% of the activity of the soluble enzyme for the hydrolysis of long-chain peptides (e.g., cheese whey proteins previously hydrolyzed with immobilized trypsin and chymotrypsin) and of the small substrate N-benzoylglycyl-l-phenylalanine (hippuryl-l-Phe). These results were much better than those achieved using the conventional support, glutaraldehyde-agarose. Amino acid analysis of the products of the acid hydrolysis of CPA (both soluble and immobilized) showed that approximately four lysine residues were linked on the glyoxyl agarose beads, suggesting the existence of an intense multipoint covalent attachment between the enzyme and the support. The maximum temperature of hydrolysis was increased from 50 degrees C (soluble enzyme) to 70 degrees C (most stable CPA-glyoxyl derivative). The most stable CPA-glyoxyl derivative could be efficiently used in the hydrolysis of long-chain peptides at high temperature (e.g., 60 degrees C), being able to release 2-fold more aromatic amino acids (Tyr, Phe, and Trp) than the soluble enzyme, under the same operational conditions. This new CPA derivative greatly increased the feasibility of using this protease in the production of protein hydrolysates that must be free of aromatic amino acids.
Assuntos
Carboxipeptidases A/química , Carboxipeptidases A/síntese química , Hidrocarbonetos Aromáticos/química , Proteínas do Leite/química , Fenilalanina/análogos & derivados , Fenilalanina/química , Engenharia de Proteínas/métodos , Hidrolisados de Proteína/síntese química , Aminoácidos/química , Quimotripsina/química , Desenho de Fármacos , Ativação Enzimática , Estabilidade Enzimática , Enzimas Imobilizadas/síntese química , Enzimas Imobilizadas/química , Concentração de Íons de Hidrogênio , Especificidade por Substrato , Temperatura , Tripsina/químicaRESUMO
To study the kinetics and the phenotype of the mast cells (MC) arising during infection with the nematode Nippostrongylus brasiliensis, monospecific cDNA probes for nine different MC proteases were used in a Northern blot analysis of RNA from the small intestine of infected rats. The expression was analyzed at four individual time points during infection, day 0 (before infection), and days 7, 12 and 16 post infection. A dramatic increase in mRNA for rat mast cell protease (RMCP)-2, the major mucosal MC protease in the rat, was observed, beginning around day 7 after infection and peaking around day 12. At day 16 the expression was already beginning to decline. An almost identical pattern of mRNA expression was detected for the RMCP-8 subfamily of rat MC proteases (RMCP-8, -9 and -10) and for two additional rat serine proteases, the chymases RMCP-3 and -4. No simultaneous increase in the proteases known to be expressed preferentially by mature connective tissue MC (RMCP-1, -6 and -7) was observed. This is consistent with our finding that the expansion of MC in the intestines of parasite-infected animals was limited, almost exclusively, to the mucosal MC population. However, a minor increase in RMCP-5 and MC carboxypeptidase A (CPA) mRNA was detected at day 12 after infection, suggesting a derivation of mucosal MC from an expanding RMCP-5- and CPA-positive population of MC precursors.
Assuntos
Mucosa Intestinal/metabolismo , Nippostrongylus , RNA Mensageiro/análise , Serina Endopeptidases/genética , Infecções por Strongylida/metabolismo , Animais , Carboxipeptidases/genética , Carboxipeptidases A , Quimases , Cinética , Masculino , Reação em Cadeia da Polimerase , Ratos , Ratos Sprague-Dawley , TriptasesRESUMO
A preparation of tubulin carboxypeptidase partially purified from bovine brain was found to contain a protein of molecular mass 30 kDa (P30) as determined by SDS-PAGE, that is recognized by a polyclonal anti-bovine pancreatic carboxypeptidase A. However, this protein is different from pancreatic carboxypeptidase A as judged by the isoelectric point and the pattern of peptides produced by trypsin digestion. The isoelectric point of P30 was similar to that found for tubulin carboxypeptidase (9 +/- 0.2). When the tubulin carboxypeptidase preparation was subjected to gel filtration chromatography under low salt concentration, P30 behaved as a protein of molecular mass 38 kDa whereas tubulin carboxypeptidase eluted at a position of 75 kDa molecular mass. However, when the chromatography was performed at relatively high salt concentration they behaved as proteins of 49 and 56 kDa, respectively. We considered that P30 may be an inactive monomeric form of the dimeric tubulin carboxypeptidase. However we can not rule out the possibility that it represents another carboxypeptidase not yet described.
Assuntos
Encéfalo/enzimologia , Carboxipeptidases/química , Carboxipeptidases/isolamento & purificação , Animais , Anticorpos , Western Blotting , Carboxipeptidases/metabolismo , Carboxipeptidases A , Bovinos , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Focalização Isoelétrica , CoelhosRESUMO
Using immunobinding and enzymatic assays we determined in rat muscle extracts the proportion of tyrosinatable tubulin, that is, tubulin that participates in the tyrosination/detyrosination cycle. We found that in muscle, in contrast with nervous tissue, practically all tubulin molecules are tyrosinatable. In the case of rat brain the non-tyrosinatable tubulin pool accounts for about 50% of the tubulin. In addition, isolectrofocusing of 14C-tyrosinated tubulin from brain and muscle extracts revealed a different composition in tyrosinatable tubulin isotypes. One of the isotypes, which in muscle accounts for 86% of the 14C-tyrosinated tubulin species, was detyrosinated by the action of tubulin carboxypeptidase faster than the rest of the 14C-tyrosinated tubulin isotypes taken in whole. In the case of brain extract, that isotype accounts for only 16% of the labeled tubulin.
Assuntos
Músculos/química , Tubulina (Proteína)/química , Tirosina , Animais , Anticorpos/imunologia , Radioisótopos de Carbono , Carboxipeptidases , Carboxipeptidases A , Microtúbulos/química , Ratos , Tubulina (Proteína)/análise , Tubulina (Proteína)/imunologiaRESUMO
It was found that the detyrosination of tyrosinated tubulin by tubulin carboxypeptidase can occur when both the enzyme and the substrate are adsorbed on nitrocellulose. This, and the use of a specific antibody that recognizes detyrosinated tubulin allowed us to localize tubulin carboxypeptidase on a nitrocellulose membrane after agarose gel electrophoresis and blotting. The method was also extended to detect pancreatic carboxypeptidase A.
Assuntos
Carboxipeptidases/análise , Colódio/química , Animais , Encéfalo/enzimologia , Carboxipeptidases A , Bovinos , Eletroforese em Gel de Ágar , Immunoblotting , Focalização Isoelétrica , Membranas ArtificiaisRESUMO
We have demonstrated that the isolated perfused rat mesenteric arterial bed (MAB) secretes peptidases capable of metabolizing bradykinin and angiotensin I. The major degradative pathway of bradykinin by enzymes found in the rat MAB perfusate was mediated by carboxypeptidase A-like activity, whereas angiotensin 1 degradation followed two main routes, one attributable to a carboxypeptidase A-like enzyme and the other to an endopeptidase. This latter enzyme seems to be a novel serine peptidase capable of releasing angiotensin II directly from both angiotensin I and renin substrate tetradecapeptide. The rat MAB perfusate was also shown to contain additional endo- and exopeptidases that might play a role in the metabolism of other vasoactive peptides. Our finding that isolated rat MAB secretes peptidases into the perfusion medium indicates that peptide processing within the microvasculature environment may be effected by enzymes besides those normally found in plasma or associated with cell membranes.
Assuntos
Peptídeo Hidrolases/isolamento & purificação , Circulação Esplâncnica , Peptídeo Intestinal Vasoativo/metabolismo , Sequência de Aminoácidos , Angiotensina I/metabolismo , Animais , Bradicinina/metabolismo , Carboxipeptidases/metabolismo , Carboxipeptidases A , Cromatografia em Gel , Endopeptidases/isolamento & purificação , Endopeptidases/metabolismo , Dados de Sequência Molecular , Peptídeo Hidrolases/metabolismo , Perfusão , Ratos , Ratos EndogâmicosRESUMO
Chicken erythrocytes, which contain a marginal band of microtubules, were used to study the influence of the aggregation state of tubulin on the post-translational incorporation of tyrosine into the alpha-tubulin subunit. We found that the incorporation of [14C]tyrosine occurs almost exclusively into the nonassembled tubulin pool. The marginal band was practically not labeled. The low incorporation into microtubules was not due to the lack of tubulin with acceptor capacity since after cold-induced disassembly, an additional amount of [14C]tyrosine could be incorporated. 14C-Tyrosinated tubulin of the nonassembled pool could not be incorporated into microtubules of the marginal band after prolonged incubation at 37 degrees C or when the marginal band was regenerated after cold-induced depolymerization. In erythrocytes, tubulin:tyrosine ligase behaved as a soluble entity when the cells were lysed under microtubule-preserving conditions.